Protein expression is never a simple task and the demand for high-quality biotherapeutic proteins continues to grow. Bottlenecks arise frequently because functional recombinant proteins are difficult to produce, thus protein expression engineers are often forced to return to the drawing board. This usually requires designing new cloning schemes, including lengthy verification and sequence analysis of the gene or protein of interest, moving a gene from one vector to another, transfecting the vector in an alternative host, re-characterizing the expressed protein or any of the above—an inefficient, time-consuming and expensive process.

Cambridge Healthtech Institute’s Seventh Annual Engineering Genes, Vectors, Constructs and Clones conference continues the tradition of applying effective engineering strategies for protein expression and production research leading to functional biotherapeutic products. Learn from seasoned, savvy researchers as they share their real-world experiences, applications and results.

Advances in DNA synthesis and sequencing are enabling new approaches for engineering biological systems. Such approaches build off of decades of broader efforts in the engineering and computational sciences to direct the design of proteins, pathways and organisms to perform desired functions. This presentation describes such approaches and how they are being applied to improved production of various target molecules.

We present an integrative Escherichia coli model that bridges the transcriptional, signal transduction and metabolic layers through a constraint-based framework. Training of this modeling over the most extensive normalized genome-scale compendium resulted in a performance highly predictive of growth and expression under various environments. We discuss how such models can be used in synthetic biology in conjunction with automated design platforms and new extensions for integration of proteomics and condition-specific predictions.

11:15 Mammalian Systems and Synthetic Biotechnology for Recombinant Protein Production

The increasing demand for recombinant therapeutic proteins highlights the need to constantly improve the efficiency, yield and quality of these products from mammalian cells. We have recently established a “mammalian systems and synthetic biotechnology” framework where omics-data-driven and hypothetical model-driven approaches are integrated to study their growth characteristics, enhance cellular performance and design novel biological products or functions. This talk presents several applications of the framework.

We have generated an expanded genetic alphabet consisting of the two natural base pairs, A–T and G–C, and a third unnatural base pair of our own design. Recently, we have demonstrated that Escherichia coli may be engineered to import the requisite unnatural triphosphates, and that the resulting six-letter DNA is efficiently replicated and transcribed within the cell. This represents the first semi-synthetic organism that can store and retrieve increased information in its genome.

We have developed a technology that harnesses the naturally abundant ion channel producing capabilities of Tetrahymena thermophila to enable high-density display of correctly folded and functional recombinant human ion channels on the Tetrahymena cell surface. A drug discovery toolbox has been developed that includes surface membrane fractions enriched in these channels and large (milligram) amounts of functionally reconstituted purified ion channels for use as immunogens for antibody discovery and/or incorporation into platforms for small molecule drug screening.

Current gene synthesis technologies allow unprecedented capability to tailor biological systems for a wide range of purposes. We describe how gene synthesis in concert with machine learning methods can be used to identify and engineer gene, pathway, genome or protein sequence variables critical for performance. Examples of application for protein expression, pathway optimization and protein engineering are discussed.

The amino acids chosen to be included in a protein expression construct can greatly affect the usability of a protein in the laboratory. Dimensions BioSciences has developed a scoring system that systemically analyzes the primary and secondary structure of a construct and determines the amino acid sequence most likely to remain soluble throughout purification and downstream experiments. Current applications and calibrations for our scoring system will be presented.

Phage display is widely used in discovery of therapeutic antibodies. A bottleneck in the screening of clones from phage display libraries is the subcloning of variable regions to mammalian vectors for expression of IgG. We developed a vector for Fab phage display that expresses multiple antibody formats in mammalian cells without reformatting, expediting the screening of clones derived by phage display.

The Strep-tag®/Strep-Tactin® system is commonly known to provide highly pure and functional proteins at reasonable cost. IBA´s latest development, Strep-Tactin XT, completes now the product portfolio by enabling stable but still reversible immobilization for novel assay applications.

Cytosolic DNA degradation plays an important role in decreasing transgene expression; however, the cleavage locations remain largely unexplored. High-throughput sequence mapping of cytosolic nuclease cleavage sites for Luciferase plasmid in HeLa cells revealed the following most common cut sites: the poly(A) region between the β-lactamase gene and the cytomegalovirus promoter, the 5’ end of the β-lactamase gene, the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter.

Recombinant protein expression is a valuable tool in many industrial applications; however, expression problems often arise. We have generated a vector suite for easy cloning and evaluation of different expression parameters affecting recombinant protein production yields. An attractive application for some recombinant proteins is their use as binding reagents. We evaluated a new library design derived from Sac7d variants (Affitins) to generate binding proteins that can work as potent protein inhibitors.

Major bacterial protein production systems use plasmids as vectors. We present new data demonstrating high levels of proteins expression without plasmids. We show how this leads to faster development times, and can substantially lower cost of production.